Many kinds of cloning vectors exist. However, no versatile cloning vector which can be functional in different microorganisms such as in Bacillus, E. coli and Pseudomonas has generally been known. Such a versatile cloning vector functional only in Streptomyces, Bacillus and E. coli (Jap. Pat. Unexam. Publ. No. 58-134100) or in Corynebacterium and Brevibacterium (ibid. No. 58-105999) is known.
An object of the present invention is to provide novel versatile cloning vectors which can be functional in Bacillus, Pseudomonas and E. coli and which can include one or more drug resistance genes in each of these microorganisms.
The importance of vectors to genetic engineering is described in Recombinant Molecules; Impact on Science and Society, Miles International Symposium Ser. No. 10, Ed. by R. F. Beers, Raven Press, New York (1977).
In E. coli, plasmid vectors such as pBR 322 have often been used. The numerous advantages of plasmid vectors like pBR 322 are as follows:
(1) a high copy number in bacterial cells;
(2) a low molecular weight and limited number of sites for restriction endonuclease cleavage provide an essential simplicity for cloning foreign DNA fragments while retaining replication activity;
(3) bacterial cell populations which contain selectable markers such as ampicillin- and tetracycline-resistance genes and the ability to easily isolate large quantities of plasmid have made plasmid vectors the most useful to date;
(4) the insertion of foreign DNA into restriction enzyme cleavage sites of a plasmid, i.e. the PstI site in ampicillin-resistant genes and BamHI, HindIII and SalI sites in tetracycline-resistant genes, transforming drug-resistance to drug-sensitivity due to breakage of the resistant genes, provides easy selection of recombinant DNA holding bacteria by selecting plasmid holding cells with one drug resistant nature, while selecting susceptibility to another drug. [F. Boliva et al., Gene, 2, 95 (1977)].
Many different types of plasmid vectors have been prepared for E. coli and other industrially useful bacteria, for example, the amylase-producing bacteria Bacillus, and the waste decomposing strain Pseudomonas. Unfortunately, however, no easily manipulable plasmid vector as compared with those in E. coli has been found in Bacillus or Pseudomonas. Plasmid replication and gene expression are restricted to and affected by background factors in transformed cells. For example, transduction of a drug resistance gene of E. coli ligated to a plasmid of Bacillus and transformed into Bacillus will replicate its recombinant plasmid, but cannot express the drug resistance element derived from E. coli. [J. Kreft et al., Mol. Gen. Genet., 162, 59 (1978); J. L. Schottel et al., J. Bacteriol., 146, 360 (1981).] Additionally, because of the lack of a Pseudomonas specific origin of replication, the E. coli plasmids pBR 322 and pACYC 184 cannot transform Pseudomonas. [Plasmid Medical, Environmental and Commercial Importance, K. N. Timmis and A. Puhler, Eds., pp 411-422].
Plasmid vectors pBR 322 or pCRI, which possess the replication origins of pMBI or ColEI, were originally non-transferable plasmids. However, they can be transferred together with a coexisting transferable and another third non-transferable plasmid. [Current Chemoth. and Immunotherapy, Proc. 12th Inter. Cong. Chemoth., Vol. 1, p. 6.] These vectors are not preferable from a safety point of view, because it may be dangerous to transfer a drug resistance gene in a vector and an unknown gene in a recombinant.
Plasmid vectors developed in E. coli are, in their replication origins, limited to ColEI, pMBI, p 15A and R 6-5. When an insertion of plural plasmid vectors in a cell is required in order to increase productivity and to investigate gene relation, there may occur incompatibility, namely such like vectors cannot coexist stably in a host cell and are eliminated.
A vector, RP-4, often used in Pseudomonas, is a large, transferable plasmid of molecular weight 38Md. It is not a preferred vector, because the genetic information required for RP-4 replication is located on a separate plasmid molecule and hence the preparation of a smaller vector plasmid is difficult. Only the plasmids RSF 1010 and Rlb 679 (5.5Md) have been reported; however, they are not preferred because insertional inactivation cannot be carried out using easily handled restriction enzymes.
Bacillus has the following advantages:
(1) it is a production strain of antibiotics butyrocin and polymyxin;
(2) it is a production strain of amylase and protease;
(3) it is an important industrial strain for exoenzymes; and
(4) it is non-parasitic in humans and therefore safe for receipt of foreign DNA.
Regrettably, vector development has not progressed as far or as quickly in Bacillus as it has in E. coli. One reason for this is that the multiple drug resistance genes of E. coli cannot be expressed hence in Bacillus, and hence resistance markers of E. coli cannot apply to Bacillus. Furthermore, the effectiveness of transformation using competent cells is much lower at 10.sup.2 -10.sup.3 transformants per 1 .mu.g of DNA as compared with that of the E. coli series at 10.sup.8 to 10.sup.12 transformants. Alternatively, Bacillus transformation by protoplast fusion improved the yield of transformants but requires time and practice, so that development of a drug marker having a preferred restriction site applicable to insertional inactivation might be delayed. Shuttle-vectors of Bacillus and E. coli have been reported [Abstract, Jap. Agr. Biol. Chem. Soc. Meeting 1980, p. 408], but unlike E. coli vectors the recombinant DNA technique for Bacillus expression of resistance against more than one kind of drug cannot be observed and so no insertion-inactivation method is used.